Sexual reproduction is prevalent in eukaryotes and plays a key role in their fitness and survival. Asexual species often arise, but regularly go extinct due, in part, to deleterious mutation accumulation and the inability to adapt to changing environments. Bdelloid rotifers are microscopic animals comprised entirely of females that reproduce asexually by parthenogenesis. Sexual reproduction and meiosis are completely unknown in bdelloids, even though related rotifers are capable of sex. Fossil evidence and molecular genetic studies suggest that bdelloids are "ancient asexuals", persisting for up to 100 million years without sex. Since meiosis is central to sexual reproduction, the central goal of this research is to determine the presence and study the evolution of meiosis-specific genes in genomes of sexual and bdelloid rotifers. Organisms possessing meiotic genes may be capable of meiotic sex. Our approach employs PCR to amplify meiosis- specific genes. We have already amplified partial sequences of four meiotic genes (MND1, DMC1, SPO11, MSH5) from bdelloid rotifers. We will survey the genomes of multiple bdelloid and related sexual species (eight in total) for the presence of these and five additional meiotic genes (MSH4, HOP1, HOP2, REC8, RDH54). This set of genes defines our "meiosis detection kit" that we will use to look for evidence of meiosis in bdelloids. Partial gene sequences will be used as probes to screen genomic (fosmid/cosmid) libraries of bdelloids and their sexual relatives (monogonont and acanthocephalan rotifers). Some libraries are available and we will construct four others. Library screens will allow us to isolate complete genes and to detect additional gene copies not amplified by PCR. Cosmid clones of meiotic genes will be used in subsequent FISH analyses to verify the presence and to document the chromosomal distribution of meiotic genes in rotifer genomes. Rigorous evolutionary analyses of rotifer meiotic genes will assess phylogenetic relationships and evolutionary rates, compared to mitotic genes and with homologs in sexual taxa. These analyses will allow functional inferences for the isolated genes. RNA expression studies using RT-PCR methods will be used to examine the functionality of these genes in sexual and bdelloid rotifers. The results from this project will either support the remarkable status of bdelloid rotifers as ancient asexuals or they will provide genetic evidence suggesting the bdelloids are capable of meiosis and possibly, sexual reproduction. Reproductive modes are crucial to the biology and population genetics of all organisms, and this is especially true for pathogens. Thus, the methods developed here have applications relevant to human health and disease;our approach to infer the presence of meiosis will be relevant to studies of parasite resistance, epidemiology, disease treatment and management.